Methods for selecting plants after genome editing

ABSTRACT

The disclosure provides methods for selecting modified plants with a mutation in a target gene and plants produced by the methods. Specifically, the disclosure provides methods comprising introducing a recombinant expression cassette encoding a genome editing protein into meristematic or germline cells of a parent plant, wherein the genome editing protein specifically recognizes a target gene; crossing or selfing the parent plant, thereby producing a plurality of progeny seeds; and selecting progeny plants grown from the progeny seeds that express a phenotype that can be selected at the intact plant level.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) to U.S.application Ser. No. 61/991,173, filed May 9, 2014, the contents ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods of conferring a desiredphenotype on a plant by specifically mutating a target gene in a plantusing a genome editing procedure.

BACKGROUND OF THE INVENTION

Recent advances in gene editing technologies have provided opportunitiesfor precise modification of the genome in many types of organisms,including plants and animals. In particular, technologies based ongenome editing proteins, such as zinc finger nucleases, TALENs, andCRISPR-Cas9 systems are advancing rapidly and it is now possible totarget genetic changes to specific DNA sequences in the genome (seee.g., Segal, et at. (2013) Annu. Rev. Genomics Hum. Genet. 14, 135-158;Sander, et at. (2014) Nature Biotech. 32, 347-355). These can be eithermutations resulting in gene knockouts or substitutions of one allele foranother. Highly efficient CRISPR-mediated precise genome modificationhas now been demonstrated in several plants. In most plants, it isnecessary to go through tissue culture to obtain modifications of thegermline. Tissue culture can be mutagenic and therefore there is thelikelihood of introducing unpredictable and undesired background,off-target mutations in addition to the desired changes. Also, itremains challenging to select for cells with the targeted genomemodification.

What is desired is a method that does not require going through tissueculture and allows the selection of modified cells without the use ofselectable transgenes. The present invention provides these and otheradvantages.

BRIEF SUMMARY OF THE INVENTION

This invention provides method for selecting modified plants with amutation in a target gene and plants produced by the methods. Themethods comprise (a) introducing a first recombinant expression cassetteencoding a first genome editing protein or protein plus oligonucleotide(e.g., a guide RNA) into meristematic or germline cells of a parentplant, wherein the first genome editing protein or protein plusoligonucleotide (e.g., a guide RNA) specifically recognizes the targetgene; (b) introducing a second recombinant expression cassette encodinga second genome editing protein or protein plus oligonucleotide (e.g., aguide RNA) into meristematic or germline cells of the parent plant,wherein the second genome editing protein or protein plusoligonucleotide specifically recognizes a gene controlling seedgermination; (c) after steps (a) and (b), crossing or selfing the parentplant, thereby producing a plurality of progeny seed (e.g., firstgeneration progeny); (d) selecting progeny plants grown from the progenyseed that express a phenotype that can be selected at the intact plantlevel. In some embodiments, this phenotype is the ability to germinateunder conditions that inhibit germination of seed which lack a mutationin the gene controlling seed germination; and (e) identifying theprogeny plants selected in step (d) that comprise a mutation in thetarget gene, thereby selecting modified plants with a mutation in thetarget gene

The recombinant expression cassettes can be introduced into the cells invitro or in planta. The method of the invention can be used withessentially any plant. In some embodiments, the parent plant is lettuce.The gene controlling seed germination can be one that inhibitsgermination at high temperatures (e.g., at or above 30° C.). Anexemplary gene for this purpose is LsNCED4 in lettuce.

A number of genome editing proteins can be used in the invention.Examples include, zinc finger nucleases, TALENs, or Cas-9 nucleasesguided by a guide RNA (gRNA). The genome editing proteins can be used tointroduce a single strand nick or a double strand break in the targetgene, which is repaired and leads to a mutation in the target sequence.The genome editing proteins can also be used to introduce a desirednucleotide sequence into the target gene by homologous recombination.

The expression cassettes can be introduced into the cells usingtechniques, including use of Agrobacterium, particle bombardment, ormicroinjection. The expression cassettes can be integrated into thegenome of the plant or the genome editing proteins can be transientlyexpressed in the cells.

The methods of the invention can be used to confer any desired phenotypeon the plants. Exemplary phenotypes include resistance to plantpathogens, resistance to stress conditions (biotic or abiotic),increased yield, or changes plant development.

DEFINITIONS

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g., leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g., bracts, sepals, petals, stamens,carpels, anthers and ovules), seed (including embryo, endosperm, andseed coat) and fruit (the mature ovary), plant tissue (e.g., vasculartissue, ground tissue, and the like) and cells (e.g., guard cells, eggcells, trichomes and the like), and progeny of same. The class of plantsthat can be used in the method of the invention is generally as broad asthe class of higher and lower plants amenable to transformationtechniques, including angiosperms (monocotyledonous and dicotyledonousplants), gymnosperms, ferns, and multicellular algae. It includes plantsof a variety of ploidy levels, including aneuploid, polyploid, diploid,haploid and hemizygous.

The term “progeny” refers generally to the offspring of selfing or across and includes direct first generation progeny (e.g., F1), as wellas later generations (e.g., F2, F3, etc).

As used herein, “transgenic plant” includes reference to a plant thatcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid, including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic.

A “non-transgenic plant” is a plant that lacks a heterologouspolynucleotide stably integrated into its genome. Such a plant maycomprise alterations of its genome (chromosomal or extra-chromosomal)that are introduced by the methods of the invention, conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

The term “expression cassette” refers to any recombinant expressionsystem for the purpose of expressing a nucleic acid sequence of theinvention in vitro or in vivo, constitutively or inducibly, in any cell,including, in addition to plant cells, prokaryotic, yeast, fungal,insect or mammalian cells. The term includes linear and circularexpression systems. The term includes all vectors. The cassettes canremain episomal or integrate into the host cell genome. The expressioncassettes can have the ability to self-replicate or not (i.e., driveonly transient expression in a cell). The term includes recombinantexpression cassettes that contain only the minimum elements needed fortranscription of the recombinant nucleic acid.

The term “constitutive” or “constitutively” denotes temporal and spatialexpression of the polypeptides of the present invention in plants in themethods according to various exemplary embodiments of the invention. Theterm “constitutive” or “constitutively” means the expression of thepolypeptides of the present invention in the tissues of the plantthroughout the life of the plant and in particular during its entirevegetative cycle. In some embodiments, the polypeptides of the presentinvention are expressed constitutively in all plant tissues. In someembodiments, the polypeptides of the present invention are expressedconstitutively in the roots, the leaves, the stems, the flowers, and/orthe fruits. In other embodiments of the invention, the polypeptides ofthe present invention are expressed constitutively in the roots, theleaves, and/or the stems.

The term “inducible” or “inducibly” means the polypeptides of thepresent invention are not expressed, or are expressed at very lowlevels, in the absence of an inducing agent. The expression of thepolypeptides of the present invention is greatly induced in response toan inducing agent.

The term “inducing agent” is used to refer to a chemical, biological orphysical agent or environmental condition that effects transcriptionfrom an inducible regulatory element. In response to exposure to aninducing agent, transcription from the inducible regulatory elementgenerally is initiated de novo or is increased above a basal orconstitutive level of expression. Such induction can be identified usingthe methods disclosed herein, including detecting an increased level ofRNA transcribed from a nucleotide sequence operatively linked to theregulatory element, increased expression of a polypeptide encoded by thenucleotide sequence, or a phenotype conferred by expression of theencoded polypeptide.

The phrase “substantially identical,” in the context of the presentinvention refers to polynucleotides or polypeptides that have sufficientsequence identity with a reference sequence to effect similarfunctionality when expressed in plants as the reference sequence. Inaccordance with one aspect of an exemplary embodiment of the invention,a polynucleotide or a polypeptide that exhibits at least 90% sequenceidentity with a reference sequence may be deemed to be “substantiallyidentical;” however, polynucleotides and polypeptides that exhibit less(even significantly less, e.g., 60%-70% or less) than 90% sequenceidentity may, in accordance with various exemplary embodiments of theinvention, be “substantially identical” to their reference sequences ifrequisite functionality is achieved. Alternatively, percent identity canbe any value from 90% to 100%. More preferred embodiments include atleast: 90%, 95%, or 99% identity as used herein is as compared to thereference sequence using the programs described herein; preferably BLASTusing standard parameters, as described below.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions, such as from 20 to600, usually about 50 to about 200, more usually about 100 to about 150,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. If no range is provided, the comparison window is the entirelength of the reference sequence. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted e.g., by the local homology algorithm ofSmith and Waterman, Adv. Appl. Math. 2:482, 1981; by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970;by the search for similarity method of Pearson and Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988; by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection.

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul, S. F. et al., J. Mol. Biol. 215:403-410, 1990.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul, S. F. et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl.Acad. Sci. USA 89:10915, 1989), alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, preferably less thanabout 0.01, and more preferably less than about 0.001.

DETAILED DESCRIPTION

The present invention provides methods of conferring a desired phenotypeon plants by specifically mutating a target gene in the plant using afirst genome editing protein to mutate the target gene and a secondgenome editing protein to mutate a second gene controlling a phenotypethat can be selected at the whole plant level (e.g., a gene thatcontrols seed germination). Plants are selected based on the presence ofa mutation in the second gene (e.g., by the ability of seed to germinateunder conditions that inhibit germination of wild type plants) andsecondarily on the presence of a mutation in the target gene.

The methods of the invention take advantage of the high rates ofsimultaneous genetic changes that occur with genome editing proteinsCRISPR-Cas9 technology and the ability to specifically modify traitsthat are controlled by a single gene and that can be selected at thewhole plant level after modification. An example of such a trait istemperature sensitivity of seed germination being determined by a singlegene. It is known that genome editing proteins introduce genemodifications at high frequency multiple times within the same cell. Insome systems up to 30% of the cells contain homozygous changes, i.e.both alleles have been modified. If multiple genes are targetedsimultaneously, up to 10% of cells have modifications in multiple genesand up to five genes have been modified simultaneously.

Generally, genome editing proteins or proteins plus oligonucleotide ofthe invention result in targeted cleavage of genomic DNA (e.g., a singlestrand nick or a double strand break). All eukaryotic organisms,including plants, repair breaks in DNA using highly conserved DNA repairmechanisms such as the non-homologous end joining or thehomology-directed repair pathways that can lead to sequencemodifications at the site of break. The non-homologous end joiningrepair pathway, for example, ligates the two broken ends, but canintroduce small insertions and/or deletions at the site of the break,which can disrupt a target gene. In some embodiments two double strandbreaks are made, repairing the double strand results in removing thematerial between the double strand breaks and rejoining the ends of thenucleotide sequence so as to excise the sequences between the doublestrand breaks. Alternatively, a homologous donor DNA, that containshomologous overlaps with DNA on either side of the double strand break,can be provided in combination with the genome editing protein.Homologous recombination at the site of the break results in thereplacement of a target sequence by the donor DNA sequence.

One of skill will recognize that the ability to engineer a trait relieson the action of the genome editing proteins and various endogenous DNArepair pathways. These pathways may be normally present in a cell or maybe induced by the action of the genome editing protein. Using geneticand chemical tools to over-express or suppress one or more genes orelements of these pathways can improve the efficiency and/or outcome ofthe methods of the invention. For example, it can be useful toover-express certain homologous recombination pathway genes orsuppression of non-homologous pathway genes, depending upon the desiredmodification.

It may also desirable to increase the odds of recovering a properlytargeted event rather than a randomly integrated event. Steps mayinclude, but are not limited to, the use of a positive-negativeselection system to reduce the recovery of non-targeted events (Teradaet at. 2004 Plant Cell Reports, 22:653-9).

The methods of the invention can be used to confer a desired phenotypeon essentially any plant. The invention thus has use over a broad rangeof agronomically important species including species from the generaAsparagus, Atropa, Aven, Brassica, Citrus, Citrullus, Capsicum, Cucumis,Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus,Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon,Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum,Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio,Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and Zea.In the case of selection for germination at high temperature, manyalthough not all, species are sensitive to thermoinhibition ofgermination, examples of crop species that will not normally germinateat temperature include but are not limited to lettuce, spinach, carrots,and celery.

Genome Editing Proteins

Any of a number of genome editing proteins well known to those of skillin the art can be used in the methods of the invention. The particulargenome editing protein used is not critical, so long as it provides sitespecific mutation of a desired nucleic acid sequence. Exemplary genomeediting proteins include targeted nucleases such as engineered zincfinger nucleases (ZFNs), transcription-activator like effector nucleases(TALENs), and engineered meganucleases. In addition, the CRISPR/Cassystem with an engineered RNA (e.g., a gRNA) to guide the nuclease(e.g., Cas-9) to the target cleavage site can be used.

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are engineered proteins comprising a zincfinger DNA-binding domain fused to a nucleic acid cleavage domain, e.g.,a nuclease. The zinc finger binding domains provide specificity and canbe engineered to specifically recognize any desired target DNA sequence.For a review of the construction and use of ZFNs in plants and otherorganisms, see Urnov et at. 2010 Nat Rev Genet. 11(9):636-46.

The zinc finger DNA binding domains are derived from the DNA-bindingdomain of a large class of eukaryotic transcription factors called zincfinger proteins (ZFPs). The DNA-binding domain of ZFPs typicallycontains a tandem array of at least three fingers each recognizing aspecific triplet of DNA. ZFPs have been identified in plants, where theyare involved in, for example, developmental regulation of various floraland vegetative organs. In plant ZFPs, zinc fingers do not generallyoccur in closely-spaced tandem arrays but may be separated by anintervening stretch of up to 200 amino acids. The binding capability ofthis class of ZFPs appears to be determined by both the zinc fingers andthe intervening amino acids, suggesting that plant ZFPs have a differentmechanism of DNA binding as compared to ZFPs derived from otherorganisms.

One of skill will recognize that a number of strategies can be used todesign the binding specificity of the zinc finger binding domain. Oneapproach, termed “modular assembly,” relies on the functional autonomyof individual fingers with DNA. In this approach, a given sequence istargeted by identifying zinc fingers for each component triplet in thesequence and linking them into a multifinger peptide. Severalalternative strategies for designing zinc finger DNA binding domainshave also been developed. These methods are designed to accommodate theability of zinc fingers to contact neighboring fingers as well asnucleotides bases outside their target triplet.

Typically, the engineered zinc finger DNA binding domain has a novelbinding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, for example, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See e.g., U.S. Pat. Nos. 6,453,242 and 6,534,261.Exemplary selection methods, including phage display and two-hybridsystems, are well known and described in the literature. In addition,enhancement of binding specificity for zinc finger binding domains hasbeen described in WO 02/077227.

In addition, individual zinc finger domains may be linked together usingany suitable linker sequences. See, U.S. Pat. Nos. 6,479,626; 6,903,185;and 7,153,949 for exemplary linker sequences. The ZFNs of the inventionmay include any combination of suitable linkers between the individualzinc fingers of the protein.

The nucleic acid cleavage domain is non-specific and is typically arestriction endonuclease, such as FokI. This endonuclease must dimerizeto cleave DNA. Thus, cleavage by FokI as part of a ZFN requires twoadjacent and independent binding events, which must occur in both thecorrect orientation and with appropriate spacing to permit dimerformation. The requirement for two DNA binding events enables morespecific targeting of long and potentially unique recognition sites.FokI variants with enhanced activities have been described. See e.g.,Guo, et at. 2010 J. Mol. Biol. 400:96-107.

Transcription-Activator Like Effector Nucleases (TALENs)

Transcription activator like effectors (TALEs) are proteins secreted bycertain species of Xanthomonas to modulate gene expression in hostplants and to facilitate bacterial colonization and survival. TALEs actas transcription factors and modulate expression of resistance genes inthe plants. Recent studies of TALEs have revealed the code linking therepetitive region of TALEs with their target DNA-binding sites. TALEscomprise a highly conserved and repetitive region consisting of tandemrepeats of mostly 33 or 34 amino acid segments. The repeat monomersdiffer from each other mainly at amino acid positions 12 and 13. Astrong correlation between unique pairs of amino acids at positions 12and 13 and the corresponding nucleotide in the TALE-binding site havebeen found. The simple relationship between amino acid sequence and DNArecognition of the TALE binding domain allows for the design DNA bindingdomains of any desired specificity.

TALEs can be linked to a non-specific DNA cleavage domain to preparegenome editing proteins, referred to as TALENs. As in the case of ZFNs,a restriction endonuclease, such as FokI, can be conveniently used. Fora description of the use of TALENs in plants, see Mahfouz et at. 2011Proc Natl Acad Sci USA. 108:2623-8 and Mahfouz 2011 GM Crops. 2:99-103.

Meganucleases

Meganucleases are endonucleases that have a recognition site of 12 to 40base pairs. As a result, the recognition site occurs rarely in any givengenome. By modifying the recognition sequence through proteinengineering, the targeted sequence can be changed and the nuclease canbe used to cleave a desired target sequence. (See Seligman, et at. 2002Nucleic Acids Research 30: 3870-9 WO06097853, WO06097784, WO04067736, orUS20070117128).

CRISPR/Cas System

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR-associated) system, are adaptive defense systems in prokaryoticorganisms that cleave foreign DNA. CRISPR loci in microbial hostscontain a combination of CRISPR-associated (Cas) genes as well asnon-coding RNA elements which determine the specificity of theCRISPR-mediated nucleic acid cleavage. Three types (I-III) of CRISPRsystems have been identified across a wide range of bacterial hosts. Inthe typical system, a Cas endonuclease (e.g., Cas9) is guided to adesired site in the genome using small RNAs that targetsequence-specific single- or double-stranded DNA sequences. TheCRISPR/Cas system has been used to induce site specific mutations inplants (see Miao et at. 2013 Cell Research 23:1233-1236).

The basic CRISPR system uses two non-coding guide RNAs (crRNA andtracrRNA) which form a crRNA:tracrRNA complex that directs the nucleaseto the target DNA via Wastson-Crick base-pairing between the crRNA andthe target DNA. Thus, the guide RNAs can be modified to recognize anydesired target DNA sequence. More recently, it has been shown that a Casnuclease can be targeted to the target gene location with a chimericsingle-guide RNA (sgRNA) that contains both the crRNA and tracRNAelements. It has been shown that Cas9 can be targeted to desired genelocations in a variety of organisms with a chimeric sgRNA (Cong et at.2013 Science 339:819-23).

Introduction of Genome Editing Proteins into Plant Cells

The genome editing protein may be introduced into the plant cell usingstandard genetic engineering techniques, well known to those of skill inthe art. In the typical embodiment, recombinant expression cassettescomprising a polynucleotide encoding a genome editing protein of theinvention can be prepared according to well-known techniques. In thecase of CRISPR/Cas nuclease, the expression cassette may transcribe theguide RNA, as well.

Such plant expression cassettes typically contain the polynucleotideoperably linked to a promoter (e.g., one conferring inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific/selective expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

A number of promoters can be used in the practice of the invention. Aplant promoter fragment can be employed which will direct expression ofthe desired polynucleotide in all tissues of a plant. Such promoters arereferred to herein as “constitutive” promoters and are active under mostenvironmental conditions and state of development or celldifferentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region.

Alternatively, the plant promoter can direct expression of thepolynucleotide under environmental control. Such promoters are referredto here as “inducible” promoters. Environmental conditions that mayaffect transcription by inducible promoters include biotic stress,abiotic stress, saline stress, drought stress, pathogen attack,anaerobic conditions, cold stress, heat stress, hypoxia stress, or thepresence of light.

In addition, chemically inducible promoters can be used. Examplesinclude those that are induced by benzyl sulfonamide, tetracycline,abscisic acid, dexamethasone, ethanol or cyclohexenol.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues suchas leaves, roots, fruit, seeds, or flowers. These promoters aresometimes called tissue-preferred promoters. The operation of a promotermay also vary depending on its location in the genome. Thus, adevelopmentally regulated promoter may become fully or partiallyconstitutive in certain locations. A developmentally regulated promotercan also be modified, if necessary, for weak expression.

Methods for transformation of plant cells are well known in the art, andthe selection of the most appropriate transformation technique for aparticular embodiment of the invention may be determined by thepractitioner. Suitable methods may include electroporation of plantprotoplasts, liposome-mediated transformation, polyethylene glycol (PEG)mediated transformation, transformation using viruses, micro-injectionof plant cells, micro-projectile bombardment of plant cells, andAgrobacterium tumeficiens mediated transformation. Transformation meansintroducing a nucleotide sequence in a plant in a manner to cause stableor transient expression of the sequence.

In some embodiments of the invention, in planta transformationtechniques (e.g., vacuum-infiltration, floral spraying or floral dipprocedures) are used to introduce the expression cassettes of theinvention (typically in an Agrobacterium vector) into meristematic orgermline cells of a whole plant. Such methods provide a simple andreliable method of obtaining transformants at high efficiency whileavoiding the use of tissue culture. (see, e.g., Bechtold et at. 1993 C.R. Acad. Sci. 316:1194-1199; Chung et at. 2000 Transgenic Res.9:471-476; Clough et at. 1998 Plant J. 16:735-743; and Desfeux et at.2000 Plant Physiol 123:895-904). In these embodiments, seed produced bythe plant comprise the expression cassettes encoding the genome editingproteins of the invention. The seed can be selected based on the abilityto germinate under conditions that inhibit germination of theuntransformed seed.

If transformation techniques require use of tissue culture, transformedcells may be regenerated into plants in accordance with techniques wellknown to those of skill in the art. The regenerated plants may then begrown, and crossed with the same or different plant varieties usingtraditional breeding techniques to produce seed, which are then selectedunder the appropriate conditions.

The expression cassette can be integrated into the genome of the plantcells, in which case subsequent generations will express the genomeediting proteins of the invention. Alternatively, the expressioncassette is not integrated into the genome of the plants cell, in whichcase the genome editing proteins is transiently expressed in thetransformed cells and is not expressed in subsequent generations.

In some embodiments, the genome editing protein itself, is introducedinto the plant cell. In these embodiments, the introduced genome editingprotein is provided in sufficient quantity to modify the cell but doesnot persist after a contemplated period of time has passed or after oneor more cell divisions. In such embodiments, no further steps are neededto remove or segregate away the genome editing protein and the modifiedcell.

In these embodiments, the genome editing protein is prepared in vitroprior to introduction to a plant cell using well known recombinantexpression systems (bacterial expression, in vitro translation, yeastcells, insect cells and the like). After expression, the protein isisolated, refolded if needed, purified and optionally treated to removeany purification tags, such as a His-tag. Once crude, partiallypurified, or more completely purified genome editing proteins areobtained, they may be introduced to a plant cell via electroporation, bybombardment with protein coated particles, by chemical transfection orby some other means of transport across a cell membrane.

The genome editing protein can also be expressed in Agrobacterium as afusion protein, fused to an appropriate domain of a virulence proteinthat is translocated into plants (e.g., VirD2, VirE2, VirE2 and VirF).The Vir protein fused with the genome editing protein travels to theplant cell's nucleus, where the genome editing protein would produce thedesired double stranded break in the genome of the cell. (see Vergunstet at. 2000 Science 290:979-82).

Selecting Plants with Desired Phenotypes

As noted above, one of the genome editing proteins plants of theinvention induces mutations in a gene that confers a phenotype that canbe selected at the whole plant level. Suitable genes that can betargeted for this purpose include genes that control seed germination(e.g., sensitivity to environmental conditions such as temperature,light, oxygen, water and the like), resistance to a pathogen,insensitivity to a chemical such as an herbicide or growth regulator,response to an environmental signal such as day length or temperature.Alternatively, the mutation may result in a visually detectable changein phenotype such as color or growth rate.

Plants typically have an upper or lower temperature limit at which seedgermination will occur. This is under genetic control. For example, itis well documented in lettuce that inhibition of germination at hightemperature is dependent on the activity of the LsNCED4 gene (see e.g.,GenBank Accession JN788925.1). This gene encodes a regulated enzyme inthe abscisic acid (ABA) biosynthetic pathway and if active the lettuceseed will not germinate at elevated temperatures (e.g., above 30° C.).If the gene is knocked out, the seed will germinate well at elevatedtemperatures. Therefore, the ability to germinate at high temperatureprovides a powerful screen for inactivation of the LsNCED4 gene. Ofrelevance to this invention, most, if not all, lettuce cultivars studiedhave an active LsNCED4 gene and therefore do not germinate at hightemperature. Consequently, this gene provides a natural selectablemarker for gene modification that is present in lettuce cultivars.Furthermore, inactivation of this gene seems to have no deleteriouseffect on the performance of the plant.

Thus, in a typical embodiment, the lettuce gene LsNCED4 can be used as atarget of one of the genome editing proteins in the methods of theinvention. The resultant seeds are germinated at a temperature that isnon-permissive for seeds with the active temperature sensitivity gene.Because the seeds result from a sexual combination of gametes, evenmodifications of only one allele will become homozygous in the nextgeneration and therefore expose the recessive phenotype. All plants thatgerminate at the elevated temperature are characterized as below formodifications at the other gene(s) targeted. This will providenon-transgenic plants carrying the targeted genome modification in agenetic background identical to the progenitor one. If necessary, theinactivated temperature sensitivity gene can be removed using a singlebackcross generation.

The invention also provides molecular assays for detecting andcharacterizing cells that have been modified by one or both of thegenome editing proteins used in the methods of the invention. One ofskill will recognize that a number of molecular assays can be used forthis purpose. These assays include, for example, nucleic acidhybridization, PCR, sequencing, and the like. Methods using highthroughput, non-destructive seed sampling for one or more markers, suchas genetic markers are well known in the art. Apparatus and methods forthe high throughput, non-destructive sampling of seeds have beendescribed.

The methods of the invention can be used to introduce mutations into anydesired target gene controlling any desired phenotype. The desiredphenotype may be enhanced plant morphology, physiology, growth anddevelopment, color, shape, consumer preference, yield, nutritionalenhancement, decreased accumulation of undesirable constituents such asheavy metals and secondary metabolites, disease or pest resistance,improved interactions with beneficial or deleterious microbes such assymbionts and microbiome constituents, or tolerance to abiotic stress.The phenotype may also be enhanced water use efficiency, enhanced coldtolerance, increased yield, enhanced nitrogen use efficiency, enhancedseed protein and enhanced seed oil, better post-harvest performance andless post-harvest losses. The desired phenotype may be enhanced yieldincluding increased yield under non-stress conditions and increasedyield under environmental stress conditions. Stress conditions mayinclude, for example, drought, shade, fungal disease, viral disease,bacterial disease, insect infestation, nematode infestation, coldtemperature exposure, heat exposure, osmotic stress, reduced nitrogennutrient availability, reduced phosphorus nutrient availability,anaerobic conditions, exposure to pollutants, and high plant density.Yield can be affected by many properties including for example, plantheight, fruit or grain size, efficiency of nodulation and nitrogenfixation, efficiency of nutrient assimilation, resistance to biotic andabiotic stress, carbon assimilation, plant architecture, resistance tolodging, percent seed germination, seedling vigor, and juvenile traits.Yield can also be affected by efficiency of germination (includinggermination in stressed conditions), growth rate (including growth ratein stressed conditions), seed size, composition of seed (starch, oil,protein) and characteristics of seed fill.

One of skill will thus be able to choose an appropriate selection methodbased upon the desired phenotype. For example, where the desiredphenotype is tolerance to a particular stress condition (e.g., drought,highly salinity, or anaerobic conditions) the methods of the inventioninclude exposure of a population of plants to the stress conditions andselecting plants that show increased tolerance. Thus, a plant with thedesired phenotype, when exposed to the stress condition, shows less ofan effect, or no effect, in response to the condition as compared to acorresponding reference or control plant (naturally occurring wild-typeplant or a plant not containing a mutation in the target gene).

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Development and Application of Whole Plant CRISPR Technology

The present invention provides a strategy of gene editing to introducebeneficial allelic variation that avoids going through tissue culture.This takes advantage of the temperature sensitivity of seed germinationbeing determined by a single gene, LsNCED4 and the high rates ofsimultaneous genetic changes that occur with CRISPR-Cas9 technology.When CRISPR-mediated gene modifications occur, they often occur withhigh frequency, multiple times within the same cell. In some systems upto 60% of the cells contain homozygous changes, i.e. both alleles havebeen modified. If multiple genes are targeted simultaneously, up to 20%of cells have biallelic modifications in multiple genes and up to fivegenes have been modified simultaneously. If LsNCED4 is active, lettuceseed will not germinate above 30° C.; however, if this gene is knockedout, the seed will germinate well at temperatures above 30° C.Therefore, the ability to germinate at high temperature provides apowerful screen for inactivation of LsNCED4. All lettuce cultivarsstudied have an active LsNCED4 gene and therefore do not germinate athigh temperatures. Consequently, this gene provides a natural selectablemarker for gene modification that is present in most (possibly all)lettuce cultivars. Furthermore, inactivation of this gene seems to haveno deleterious effect on the performance of the plant. Modification ofmeristematic cells in the shoot apex will provide non-transgenic plantscarrying the targeted genome modification in a genetic backgroundidentical to the progenitor one.

To practice the invention, meristematic cells in the shoot apex ofintact plants are modified using the CRISPR-Cas9 system. Multiple genesare targeted simultaneously. One is LsNCED4; during the testing of thesystem, the second gene is the UidA gene and the recipient plant will betransgenic expressing the UidA (GUS) gene, so that the frequency ofsingle and double gene knockouts can be evaluated. Standard componentsare used, including the gene encoding Cas9 that has been codon optimizedfor expression in plants fused to the SV40 nuclear localization signalexpressed from an apex-specific promoter such as that of the ArabidopsisINCURVATA2 gene Jiang, et al. (2103) Nucl. Acids Res. doi:10.1093/nar/gkt780; Nekrasov, et at. (2103) Nat. Biotechnol. 31, 691-69.Shan, et at. (2013) Nat. Biotechnol. 31, 686-688; Xie, et at. (2013)Molecular plant. 6, 1975-1983). To avoid off-target cleavage CRISPRdesign tools that optimize the seed sequences are used; only seedsequences without any hits to the lettuce genome are used. A singleguide RNA consisting of NGG 5′ to 20 bases from the target gene plus PAMand tracrRNA sequences is expressed from the Arabidopsis U6 promoter sothe RNA will be retained in the nucleus.

The constructs are tested using infiltration with Agrobacterium andmicroprojectile bombardment of the apices of ˜month-old plants toprovide transient gene expression in the shoot primordia. Plants aregrown to maturity and allowed to self. The resultant seeds aregerminated at 30° C. that is non-permissive for seeds with an activeNCED4 gene. Because the seeds result from a combination of gametes, evenmodifications of only one allele will become homozygous in the nextgeneration and therefore expose the recessive phenotype. Plants thatgerminate at the elevated temperature are selected and characterized forGUS expression as well as by PCR and sequencing to characterize themutations occurring in the NCED4 and UidA genes. If no gene editingevents are detected, the constructs are validated using conventionalAgrobacterium-mediated transformation via tissue culture prior toattempts to optimize the shoot treatments.

Gene replacement protocols can be developed utilizing lines for whichinactivated LsNCED4 has segregated away from the inactivated UidA gene.Constructs will again target LsNCED4; however the Cas9 plus guide RNAconstruct will be co-delivered with a functional UidA gene using TRVdelivery (Baltes, et at. (2014) Plant Cell. 26:151-63).

Example 2 Genome Engineering to Generate Novel Chromosome Blocks soCombinations of Desired Traits can be Selected for as a Single MendelianUnit

The CRISPR-Cas9 system also provides the opportunity to generatemultiple chromosome breaks in the genome and induce directed chromosomerearrangements (Lee, et at. (2012) Genome Res. 22, 539-548; Qi et al.,(2013) G3. 3,1707-15). As an increasing number of chromosome regions arecharacterized as being advantageous it becomes increasingly difficult togenerate and maintain ideal combinations of genes. One option is togenerate a cassette of transgenes and generate a transgenic line; this,however, generates regulatory hurdles. Another option is to engineer thegenome to place advantageous segments in proximal chromosomal positionsso that they will be inherited as a single Mendelian unit with littlerecombination. This should have reduced regulatory hurdles compared totransgenic lines and will also allow manipulation of complex loci suchas QTLs without complete molecular characterization.

Nucleases are designed to make double-strand breaks in the arms of twochromosomes to induce balanced translocations. Such translocations areexpected, since similar simultaneous cleavages have been shown to induceup to 5 Mb deletions, duplications, and inversions. Loss of entirechromosomes can be induced by insertion of a thymidine kinase gene andappropriate selection pressure (Li et al., 2012 Nat. Biotechnol. 31,688-691). We have similarly observed large deletions and inversionsusing CRISPR/Cas systems. To encourage translocation betweennon-homologous partners, polymorphisms are included in the target sitesso that cleavage is favored on only one of the two homologs.Simultaneous cleavage of two non-homologous chromosomes will lead, insome portion of treated cells, to translocations by non-homologousend-joining repair. Such translocation can be identified by PCRamplification across the unique translocation junction, and validated bycytogenetic and FISH analysis. Further rounds of targeted translocationproduction are used to assemble the desired genomic regions in closephysical proximity to segregate as single Mendelian units.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method for selecting modified plants with a mutation in a targetgene, the method comprising a) introducing a first recombinantexpression cassette encoding a first genome editing protein intomeristematic or germline cells of a parent plant, wherein the firstgenome editing protein specifically recognizes the target gene; b)introducing a second recombinant expression cassette encoding a secondgenome editing protein into meristematic or germline cells of the parentplant, wherein the second genome editing protein specifically recognizesa gene controlling seed germination; c) after steps (a) and (b),crossing or selfing the parent plant, thereby producing a plurality ofprogeny seed; d) selecting progeny plants grown from the progeny seedthat are capable of germinating under conditions that inhibitgermination of seed which lack a mutation in the gene controlling seedgermination; e) identifying the progeny plants selected in step (d) thatcomprise a mutation in the target gene, thereby selecting modifiedplants with a mutation in the target gene.
 2. The method of claim 1,wherein the meristematic cells are in an apical meristem.
 3. The methodof claim 1, wherein the parent plant is lettuce.
 4. The method of claim1, wherein the gene controlling seed germination inhibits germination athigh temperatures.
 5. The method of claim 4, wherein the genecontrolling seed germination inhibits germination of seeds at or above30° C.
 6. The method of claim 5, wherein the gene is LsNCED4.
 7. Themethod of claim 1, wherein the step of selecting progeny plants iscarried out using first generation progeny.
 8. The method of claim 1,wherein the first or second genome editing protein is a zinc fingernuclease.
 9. The method of claim 1, wherein the first or second genomeediting protein is a TALEN.
 10. The method of claim 1, wherein the firstor second genome editing protein is a Cas-9 nuclease guided by a guideRNA (gRNA).
 11. The method of claim 1, wherein the first or secondgenome editing protein introduces a single strand nick or a doublestrand break in the target gene.
 12. The method of claim 1, wherein thefirst genome editing protein is used to introduce a desired nucleotidesequence into the target gene by homologous recombination.
 13. Themethod of claim 1, wherein the first or second recombinant expressioncassette is introduced into the cells using Agrobacterium.
 14. Themethod of claim 1, wherein the first or second recombinant expressioncassette is introduced into the cells using particle bombardment. 15.The method of claim 1, wherein the first or second recombinantexpression cassette is introduced into the cells using microinjection.16. The method of claim 1, wherein the first or the second recombinantexpression cassette is transiently expressed in the cells.
 17. Themethod of claim 1, wherein the first and second expression cassettes areintroduced into the plant in planta.
 18. The method of claim 1, whereinthe mutation in the target gene confers resistance to a plant pathogen.19. The method of claim 1, wherein the mutation in the target geneconfers resistance to stress conditions.
 20. The method of claim 1,wherein the mutation in the target gene increases yield.
 21. The methodof claim 1, wherein the mutation in the target gene changes plantdevelopment, plant color, or sensitivity to a chemical.
 22. A plant madeby the method of claim 1.